Several new classes of luminescent metal ion substitutional probe experiment for the study of the structure and function of calcium-binding proteins and nucleic are developed and exploited. Trivalent lanthanide ions are known to substitute isomorphously at calcium(II) and magnesium(II) binding sites in macromolecules very often with the retention of biological activity. Use of the intense visible radiation from a flash-lamp-pumped dye laser or a pulsed nitrogen laser pumped dye laser allows the ready observation on dilute solutions of emission from thhe ordinarily weakly luminescent trivalent lanthanide ions. Measurement of the excited state lifetimes of these ions (principally europium(III) and terbium(III) in both H2O and D2O solution provides a direct measure of the number of water molecules coordinated to the metal ion. Lifetime measurements also monitor inter-ionic (lanthanide to transition metal and inter-lanthanide) Forster-type energy transfer which permits the estimation of distance between metal ion binding sites in macromolecules. Characterization of individual distinct binding sites is made possible by time-resolved europium(III) excitation spectroscopy, a technique recently developed by us. This method involves the observation of the 7FO-5DO Transition between nondegenerate states by scanning the excitation laser source frequency and monitoring emission from the 5DO level. Different europium(III) binding sites give rise to distinct excitation peaks, each characterized by its excited state lifetime. Luminescence emissoions will be examined under high resolution to probe the metal ion coordination site. The flouorescent amino acid or nucleotide base to lanthanide ion energy transfer which sometimes sensitize lanthanide emission will be investigated. Among the macromolecules we intend to probe by our techniques are: thermolysin, parvalbumin, alpha-amylase, phosphoglycerate kinase, prothrombin, troponin C, phospholipase A2, immunoglobulin, Ca(II)-ATPase, Ca(II)-dependent modulator protein, staphyloccocal nuclease and t-RNA.